Comparative analysis of thymidylate synthase at the protein, mRNA, and DNA levels as prognostic markers in colorectal adenocarcinoma.

BACKGROUND AND OBJECTIVES: To determine the best routinely available molecular methodology for assessing thymidylate synthase (TS) as a prognostic marker in colorectal adenocarcinoma, TS was examined at the protein, mRNA, and DNA levels. Direct comparison of these routinely available assays has not been systematically studied across a large colon cancer patient cohort with long-term follow-up. METHODS: We studied 150 surgically resected colorectal adenocarcinoma patients who received postoperative 5-Fluorouracil (5-FU) chemotherapy. TS immuunohistochemistry and real-time quantitative RT-PCR and PCR genotyping on patient-matched tumor and normal tissues were performed. RESULTS: Surprisingly, mRNA values in normal tissue varied from 0.11 to 62.0 and significantly correlated with mRNA values of matched tumor tissues. Although higher tumor/normal ratios of mRNA expression trended toward poorer patient survival, neither this nor TS immunohistochemistry results were statistically significant predictors. TS tumor genotype was generally concordant with matched normal tissues. Further, the 2R/3R genotype of 5'-TSER was significantly correlated with poorer patient survival (P = 0.0249) and was also an independent prognostic marker on multivariate analysis. CONCLUSION: TS genotyping on paraffin-embedded fixed tissues proved to be the most useful method for prediction of outcome of 5-FU treatment in patients with colorectal adenocarcinoma.

Factors affecting the performance of different long terminal repeats in the retroviral vector.

The long terminal repeat (LTR) of retrovirus contains the nucleotide sequences that control gene expression. Although several different LTRs have been used in the context of retroviral vector, the activity of the various LTRs has not yet been systematically compared for their level of gene expression. We evaluated the effect of four different LTRs on gene expression using luciferase, stem cell factor, and enhanced green fluorescence protein as reporter genes. LTRs tested in this study were derived from Moloney murine leukemia virus, myeloproliferative sarcoma virus, murine stem cell virus, and spleen focus-forming virus. It was found that the level of gene expression is affected by not only LTRs but also the transgenes and the cell types in which gene expression occurs. Furthermore, the presence of other nucleotide sequences such as the internal ribosome entry site (IRES)-neo cassette could also significantly affect gene expression. Our results suggested that the LTR should be chosen carefully, more or less on an empirical basis.